1. Field of the Invention
The present invention relates to diagnostics, more specifically to the assay of enzyme activity, using a chemiluminescent reaction (a chemical reaction that results in the emission of light). The particular chemiluminescent reaction is that between a 2,3-dihydro-1,4-phthalazinedione (DPD), especially luminol or isoluminol, with an oxidant, especially hydrogen peroxide, and a peroxidase enzyme which catalyses the oxidation of the DPD by the oxidant. The oxidation is accompanied by the emission of light, thus generating a signal by which the enzyme activity can be assayed (detected or measured).
2. Description of the Prior Art
Enzymes are extensively used as labels in ligand-binder assays (e.g., immunoassay, DNA hybridization assay) and the signal amplification inherent in an enzyme catalyzed reaction confers great sensitivity on the assay. Chemiluminescent reactions are very sensitive and have been successfully used to measure enzyme activity with improvement in the detection limit for the enzyme over conventional enzyme assays L. J. Kricka, and T. P. Whitehead, Journal of Pharmaceutical and Biomedical Analysis, 5, 829-833 (1987).
Alkaline phosphatase can be measured chemiluminescently using the phosphate substrates disclosed in European Patent Application Publication No. 254051A and by Bronstein et al., Journal of Bioluminescence and Chemiluminescence, 4, 99-111, (1989). The enzyme cleaves the phosphate from the adamantly 1,2-dioxetane phosphate to produce a dephosphorylated intermediate which decomposes with the emission of light. A bioluminescent assay for this enzyme is also possible using firefly luciferin-O-phosphate (PCT Application Publication WO 87/02667). The enzyme cleaves the phosphate group and liberates firefly luciferin, which, unlike the luciferin phosphate, is a substrate in the bioluminescent reaction catalyzed by firefly luciferase: ##STR1##
Peroxidase activity can be measured chemiluminescently using luminol as a substrate and the analytical utility of this reaction is significantly improved by the addition of certain enhancers, especially certain phenols, U.S. Pat. No. 4,598,044 and British Patent Application 2205945A or amines (U.S. Pat. No. 4,729,950), all owned by National Research Development Corporation.
It has been a problem that such enhanced chemiluminescent assays require the use of a peroxidase enzyme and are therefore primarily of use in assays in which the ligand or its binding partner are peroxidase-labelled. It would be desirable to have a chemiluminescent assay in which the ligand or binding partner can be labelled with a different enzyme, especially alkaline phosphatase.
Further prior art is mentioned after the "Summary of the invention", without which its context would not be clear.